In uninfected cells, RNA is transcribed from DNA, processed, and then transported out of the nucleus and translated into protein. In cells infected with HIV-1, the viral RNA genomes must be exported out of the nucleus without being processed so they can be packaged into new viral particles. To do this the cell must bind its own RNA genome from among the host RNA in the nucleus. This is achieved using the HIV-1 Rev protein that recognizes a Rev response element (RRE) in the viral RNA. Once bound to RRE, Rev self-associates and binds other host proteins, forming a multiprotein-RNA complex that is exported from the nucleus. Our current studies are directed at describing the molecular details of this complex. In addition to contributing fundamental information on the mechanism of viral replication, these studies may highlight points of vulnerability that may be suitable targets for therapeutic intervention including Rev itself. A picture of how Rev binds to RRE has come from our previous structural studies of both Rev and the RRE. The structure of the N-terminal half of dimeric Rev (the region involved in RNA interaction) was solved for first time by using an antibody fragment (Fab) as a crystallization chaperone. The RRE RNA forms an A shape with one leg shorter than the other. The legs are about 55 Angstrom part and position the known binding sites for Rev on either arm of the A. The higher affinity binding site is on the lower part of the short arm and the lower affinity site is on the lower part of the longer arm, placing them about 55 Angstrom from each other. This spacing matches the interaction domains of the Rev dimer that are also about 55 Angstrom apart. Once bound to RRE, Rev oligomerizes forming a complex that engages with the host nuclear export machinery. The oligomerization of Rev on RRE is essential for formation of an active nuclear export complex. To study this protein association, filaments were generated from the soluble Rev dimers and in collaboration with Laboratory of Structural Biology (NIAMS), their structure was determined by high resolution electron microscopy incorporating X-ray data from the N-terminal domain of Rev dimers. Our data revealed a third interface between Rev which offers an explanation for how the arrangement of Rev subunits adapts to the A-shaped architecture of the RRE in the export-active complexes. Also, the structures contained additional density indicating that C-terminal domains (CTD) become partially ordered in the context of filaments. This is the first time structural information on the Rev CTD has been acquired as this domain is disordered in the crystals used for X-ray determinations (study published in Structure, 2016). Further studies are required to determine in more detail the structure of the export-competent ensemble to expand our understanding of HIV-1 Rev's key role in the nuclear export of viral mRNA. Using the classic biochemical approach of divide and conquer, we have focused first on Rev RNA interactions. Using a shortened and non-polymerizing form of Rev that incorporates amino acid residues 1-93 (wild type Rev is 1-115) and is further stabilized with a single chain variable fragment (scFv) antibody, we have prepared complexes with various RNA preparations corresponding to regions of the RRE. Also we have used RNA aptamers, which are RNAs that fold into 3-dimensional conformations that bind to their targets (in this case Rev). Aptamers that bind with higher affinity than Rev-binding sites on RRE have potential anti-HIV activity. We have recently determined a high resolution structure by X-ray crystallography of Rev with a high affinity binding aptamer. In this structure, dimeric Rev bridges two discontinuous aptamers, suggesting when it binds to RRE the Rev dimer is binding two RNA sequences co-localized by the RNA conformation. Using the Rev 1-93 - scFv as a proven crystallization platform we are extending structural studies to solve interactions with RRE and other high affinity aptamers. Other structural studies using cryo-EM and X-ray crystallography (collaboration with NIAMS LSB) of the larger complexes involving CRM1, RanGTP and RRE are also on-going. The crystallization of Rev complexed with other cellular proteins that have been identified as specifically binding Rev, including Nap1, B23 and tubulin, have been made and structural studies are proceeding. The antibody fragment (Fab) used for stabilizing Rev for structural studies was derived from a phage display antibody library. This chimeric antibody (human framework and rabbit variable domains), expressed in bacteria, was humanized and was effective by binding to Rev with a very high affinity, thereby preventing its oligomerization, which as mentioned above is required for its function. In previous work (from 2014) we showed that this antibody had anti-HIV-1 activity. We also showed that cyclic peptides (up to 12 amino acids long) from the antibody variable regions (CDRs) could bind to Rev but we have not yet shown whether they also have anti-HIV-1 activity. In addition, we are attempting to co-crystallize the peptides with Rev in order to obtain high-resolution structures of the complexes, which may help design or model low-molecular weight mimics with improved (stronger) binding to Rev. In a parallel approach to targeting Rev, we are using the fact that polymerization or self-association of Rev is required for function and hence is a target for drug screening. As a first step we are developing assays that can be used to measure Rev self-association and then applied to high-throughput screening where compounds that block Rev-Rev interactions can be rapidly identified. To develop robust assay we have engineered Rev to include site-specific cysteine residues for introducing site -specific fluorescent probes, which will allow sensitive monitoring.